The present invention relates to a method of recovering enzymatically active tissue plasminogen activator (tPA) from a solution such as a culture medium or cell extract, using a chromatography resin. The invention is based on the discovery that a type of resin developed originally to selectively bind immunoglobulins binds tPA with great affinity, and therefore is useful for the commercial, large scale purification of tPA from tPA-containing solutions.
TPA is an enzyme which has the capacity to bind fibrin, and to catalyze the conversion of inactive plasminogen into the fibrinolytic enzyme, plasmin. Plasmin, in turn, degrades fibrin, a major component of blood clots, or thromboses. Therefore, tPA is a useful fibrinolytic agent for instigating the cascade mechanism which disrupts potentially fatal thromboses.
Native tPA is a glycoprotein having a molecular weight of about 66,000 daltons. It can be isolated from human blood as a single chain polypeptide, or as a dipeptide, consisting of one heavy (or A) chain disulfide bonded to one light (or B) chain, the B chain having the proteolytically active site. The single chain species may be preferred in pharmaceutical preparations over the two chain species. Further, the single chain form is believed to be the circulating species in vivo which is converted to the two chain form upon cleavage at the site of the fibrin clot, to which it binds.
Because of its extremely low concentration in the blood and tissue extracts, it is not economically feasible to purify tPA from these biological fluids for commercial use. In addition, circulating tPA has short-lived fibrinolytic activity, as it is rapidly cleared from the blood by the liver, and is readily inactivated by circulating serum proteases.
In an attempt to obtain clinically valuable amounts of tPA, cells which normally produce tPA, such as embryonic and human placenta-derived cell lines (e.g., U.S. Pat. Nos. 4,505,893 and 3,904,480), kidney and lung cells (e.g., U.S. Pat. No. 4,317,882), and human melanoma (e.g., EPO No. 0041766) have been cultured. To enhance the level of tPA synthesis by these cells, various nutritive inducers such as fumaric acid, malic acid, succinic acid, and/or glycolic acid (U.S. Pat. No. 4,328,314) and antimitotic agents such as colchicine, or podophylotoxin (U.S. Pat. No. 3,904,480) have been added to the culture media.
Alternatively, with the advent of recombinant DNA technology, cell lines which have been genetically engineered to produce large amounts of tPA have been developed and cultured. For example, transformed prokaryotic cell lines such as E. coli are said to synthesize and intracellularly deposit unglycoslyated tPA (e.g., EPO No. 0093619, EPO No. 0041766 and EPO No. 0196920). However, tPA production by recombinant means has been most successful in eucaryotic hosts such as mammalian cell lines (e.g., WO No. 86/05514) which are capable of the extensive posttranslational modifications characteristic of enzymatically active tPA.
The expressed tPA must then be recovered in active form from the cell extract or liquid growth media into which it has been secreted. A number of procedures are known by which such a recovery can be achieved including affinity chromatography, selective extraction, electrophoresis, and immunological methods. Recovery from culture media is a significant undertaking in that such media typically contain many other unrelated proteins, some of which having proteolytic activities. For example, serum-supplemented media are known to contain plasmin and other serum proteases which readily degrade tPA. Known purification procedures do not necessarily protect tPA from proteolytic degradation, although attempts have been made to do so by the addition of a metal chelating agent (e.g., U.S. Pat. No. 4,317,882) or various protease inhibitors (e.g., PCT No. 8601831) which offer at best only partial protection. Accordingly, known recovery methods are most effective when used to isolate tPA from serum-free solutions. However, most of these known methods are inefficient, and some introduce potentially toxic elements. In addition, purification procedures employing immunoaffinity chromatography may be quite costly when scaled up to meet commercial needs.
Therefore, for commercial quantitites of single chain tPA to be produced in an undegraded, enzymatically active form, large scale purification procedures are required which effectively and efficiently separate it from media before much of it is cleaved.
Accordingly, it is an object of the invention to provide a rapid, simple, and commercially viable method of isolating tPA from a tPA-containing solution. Another object is to provide a method of purifying enzymatically active single chain tPA from culture media. Yet another object is to provide a method of recovering enzymatically active tPA substantially free of other nonrelated proteins from a tPA-containing solution. It is also an object of the invention to provide a method of separating tPA from other proteins present in serum-supplemented media. It is a further object to provide a method of recovering enzymatically active single chain tPA in amounts useful for the large scale commercial production of pharmaceutical formulations containing tPA.